996 diode array detector

1 mg/ml PksH and/or PksI, or mutants thereof, was incubated at 37 °C overnight with 1 mg/ml of 3-hydroxyl-3-methylglutaryl-CoA (HMG-CoA) or an N-acetylcysteamine derivative of HMG (HMG-SNAC) in a buffer containing 20 mM Tris–HCl, 150 mM NaCl, 10% glycerol, pH 7.5. The protein was precipitated with 2 volumes of acetonitrile and removed by centrifugation (20 min 13,000 rpm). The acetonitrile was removed by Speed-Vac and remaining substrate/product redissolved in the original volume of water. Assay samples were analyzed by LCMS using a Waters 2,767 HPLC linked to a Waters ZQ mass spectrometer. SNAC derivatives were separated on a Kinetex 2.6 µm, C18, 100 Å (Phenomenex) at 1 ml/min, using a 10 min gradient from 10 to 90% acetonitrile with the addition of 0.05% formic acid and detected using a Waters 996 diode array detector between 210 and 400 nm and the mass spectrometer scanning an m/z range between 150 and 600 Da. CoA derivatives were analysed using a similar method, except that the aqueous buffer was water with 25 mM ammonium acetate and 0.5% acetic acid, and the m/z range set to 750–950 Da.

The identification of the predominant active compounds was performed via high-performance liquid chromatography (HPLC) [45 (link)]. A Waters 2695 chromatographic system with a Waters 996 diode array detector and an ACE 5C18 chromatography column (250 × 4.6 mm) was used. The data were processed using Empower 2 Chromatography Data Software. The eluent system consisted of 100% acetonitrile and 1% trifluoroacetic acid. The elution program was used as presented in Table 1, with an injection volume of 10 µL, a mobile phase flow rate of 1 mL/min, a flow time of 81 min, and a column temperature of 25 °C. The active polyphenols in testing samples were identified, evaluating the retention time of the analytes and reference substances present, as well as the UV absorption from 300 to 360 nm. The reference compounds were salicin (R² = 0.9999), p-coumaric acid (R² = 0.9999), caffeic acid (R² = 0.9999), vanillic acid (R² = 0.9999), cinnamic acid (R² = 0.9999), ferulic acid (R² = 0.9999), chlorogenic acid (R² = 0.9999), apigenin (R² = 0.9999), galangin (R² = 0.9998), pinobanksin (R² = 0.9999), and pinocembrin (R² = 0.9998). The extracts were diluted 10 times with 70% ethanol (v/v). The results are presented as the mean of three measurements, p < 0.05.

LCMS data were obtained with either (LCMS method 1): a Waters 2795HT HPLC a Phenomenex Kinetex column (2.6μ, C₁₈, 100 Å, 4.6 × 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C₅ 300 Å) eluted at 0.9 mL min⁻¹, with a Waters 996 Diode Array detector between 200 and 600 nm and a Waters ZQ mass detector operating simultaneously in ES⁺ and ES⁻ modes between 100 and 650 m/z; or (LCMS method 2) a Waters 2767 sample manager connected to Waters 2545 pumps and SFO, a Phenomenex Kinetex column (2.6μ, C₁₈, 100 Å, 4.6 × 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C₅ 300 Å) eluted at 1.0 mL min⁻¹, with a waters 2998 Diode Array detector (200–600 nm) and Waters 2424 ELSD and Waters SQD-2 mass detector operating simultaneously in ES⁺ and ES⁻ modes between 100 and 650 m/z. Solvents were: A, HPLC-grade H₂O containing 0.05% formic acid; B, HPLC-grade MeOH containing 0.045% formic acid; and C, HPLC-grade CH₃CN containing 0.045% formic acid. The gradient was as follows: 0 min, 10% C; 10 min, 90% C; 12 min, 90% C; 13 min, 10% C; and 15 min, 10% C.